CA2961883A1 - Combined enterotoxigenic escherichia coli and campylobacter jejuni recombinant construct - Google Patents

Abstract

The inventive subject matter relates to a construct comprising antigens derived from multiple enterobacteria including Campylobacter jejuni capsule polysaccharide polymer, enterotoxigenic Escherichia coli recombinant polypeptide construct and lipopolysaecharide from Shigella spp.. The subject invention also relates to a method of inducing an immune response utilizing the inventive composition.

Description

COMBINCD ENTEROTOXIGENIC ESCHERICHIA COLI AND
CAMPYLOBACTER JEJLINI RECOMBINANT CONSTRUCT
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part to U.S. No.nprovisional application 11/340,003, filed January 10, 2006, which claims priority to U.S. Provisional application 60/642,771 filed January 11, 2005, and a Continuation-in-Part to U.S.
Nonprovisional application 11/524,057 filed September 20, 2006, which claims priority to U.S.

Provisional application 60/722,086, filed September 21, 2005, and a Continuation-in-Part to U.S. Nonprovisional Application 14/048,264, filed October 8, 2013, which claims priority to U.S. Provisional application 61/727,943, filed November 19, 2012, the contents of which are herein incorporated by reference. This application also claims priority' to U.S. Provisional application 62/054,454, filed 24 September 2014, U.S.
Provisional application 62/127,927, filed March 4, 2015, U.S. Provisional application 62/165,301, filed May 22, 2015, U.S. Provisional application 62/127,935, filed March 4, 2015, and U.S. Provisional application 62/075,399, filed November 5, 2014, the contents of which are herein incorporated by reference, BACKGROUND OF INVENTION
Field of the Invention

[0002] The inventive subject matter relates to a recombinant construct against enterotoxigenic Escherichia coil and Campylobacter jejuni comprising a c.:ornbined anti-ETEC recombinant polypeptide construct and C. jejuni campsule polysaccharide.

Description of Related Art [00031 Enterotoxigenic Echerichia coli (ETEC), Shigella, spp. and Campylobacter jefuni (CI) are major causes of bacterial diarrhea worldwide. Both pathogens are a serious health threat to western travelers and young children in resource-limited countries, making them apt target populations for a single or dual pathogen vaccine against ETEC
and CJ. No FDA-licensed vaccines are available for either pathogen.
[0004] ETEC causes an estimated 210 million cases of diarrhea and 380,000 deaths annually among infants and young children. Moreover, ETEC is the most common cause of travelers' diarrhea. ETEC causes diarrhea ranging in severity from mild illness to severe cholera-like purging. There are two major virulence factors, adhesive fimbriae, dubbed colonization factors (CFs), and eriterotoxins, Surface-expressed CFs, consisting of complex protein heteropolymers, mediate adherence to the small intestinal epithelium to initiate colonization within this privileged host niche. ETEC produce one or both of two different enterotoxins, a heat-labile (LT) and a heat-stable enterotoxin (STE). LT and STE intoxicate epithelial cells, resulting in fluid and electrolyte secretion and clinical diarrhea. LT is highly immunogenic and a potent adjuvant, while STI is a small, poorly immunogenic peptide.
[0005] Prevalent CFs and a non-toxic form of the LT (or its congener cholera toxin (CT)) have been the focus for several strategies to develop an ETEC vaccine. Such antigens have been used individually or bundled as components of a whole-cell killed vaccine, live vaccines vectored by attenuated ETEC or other enterobacterial species (e.g., Shigella and Vibrio cholerae 01), and purified protein vaccines. one has yet been shown to confer sufficiently high and broad levels of protection. The weight of evidence from clinical trials indicates that anti-LT immunity confers short-term protection against LT-producing ETEC. There is also evidence to show that certain CFs function as protective antigens.
There are, however, significant challenges for ETEC vaccine development. For one, about half of all ETEC express only STI, for which anti-LT immunity is not thought to be effective, thus necessitating anti-CF or anti-bacterial irmnunity. Also, the diversity of ETEC CFs poses issues for achievement of sufficiently broad coverage with inclusion of a realistic number of CFs.
SUMMARY OF THE INVENTION
[00061 The invention relates to an im_munogenic construct comprising a polypeptide construct expressing enterotoxigenic Escherichia co li (ETEC) fimbrial subunits combined with a Campylobacterjejuni capsule polysaccharide or Shigella spp lipopolysaccharide (LPS).
[0007] :11-1 a preferred embodiment, one or more Camplobacterjejuni capsule polysaccharides are conjugated to one or more Escherichia coli enterotoxigenic recombinant poiypeptide constructs. In another embodiment, Shigella LPS is conjugated to the ETEC poly-peptide construct.
[0008] Campylobacgerjejuni is associated with induction of Guillain-Barre Syndrome (GS), a post-infectious polyneuropathy that can result in paralysis. The association is due to molecular mimicry between the sialic acid containining-outer core of the lipooligosaccharide (L)S) and human gangliosides (5, 6, 89, 91). Thus, antibodies generated against LOS cores result in an autoim.mune response to human neural tissue.

3.

Use of capsule polysaccharide from C. jejuni can induce an immune response without the possible induction of Guillain-Barre Syndrome.
[0009] In a preferred embodiment, the composition comprises an ETEC
recombinant polypeptide constrict design wherein major or minor subunits, derived from the same ETEC fimbrial type, are connected, via polypeptide linkers, and stabilized by donor strand complementation. The C-terminal most ETEC major subunit is connected, via a linker, to a donor strand region from an ETEC major subunit, which can be either homologous or heterologous to the C-terminal major subunit. The immunogenic, composition can comprise a whole or an immunogenic fragment, containing a donor 13 strand region, of the ETEC firnbrial major or minor subunits. In some construct examples, in order to avoid inadvertent association of subunits, especially in subunits to each other, major ETEC fimbrial subunits can contain an N-terminal deletion of 14 to 18 amino acids.
[0010] Int another embodiment one or more of the above constructs are connected, via a polypeptide linker, to form a multipartite fusion construct, wherein the subunits derived from multiple iimbrial types are expressed. In this embodiment, the fimbrial subunits can be derived from any ETEC fimbrial type, including, but not limited to: ETEC
class 5 fimbriae type, including class 5a, 5b or 5c; ETEC CS3; and ETEC CS(.
[00111 The embodied multipartite construct can contain a deletion of the N-terminal region of one or niore fimbrial subunits to avoid undesirable associations with other monomers or multimers and to remove reduce amino acid sequence length between polypeptides to reduce the protease cleavage.

4 [00121 DNA encoding the ETEC recombinant polypeptide construct can be used to express a polypeptide for attachment to C. jejuni or Shigeila LPS. As such, an object of the invention also includes a use of a construct for immunizing mammals, including humans, by a composition comprising antigens from rmiltiple bacterial species, including ETEC. C.jejuni and Shigella strains. The embodied use comprises one or rnore priming administrations of the combination construct. The priming dose can be subsequently followed by one or more boosting doses.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Illustration of inventive construct design wherein major or minor subunits, derived from the same ETEC fimbrial type are connected, via polypeptide linkers and stabilized by donor strand complementation. The construct can contain a deletion of the N-terminal region of the N-tenrninal subunit. This feature prevents undesirable association with other monomers or multimers. The C-terminal subunit is stabilized by a donor 0 strand, connected to the subunit via a polypeptide linker, wherein the donor strand is either derived from a homolgous subunit, which is defined as a subunit that is the same as the subunit the donor strand is stabilizing or from a heterologous subunit, defined as derived from a subunit that is different still from the same fimbrial type.
FIG. 2 illustrates a multipartite construct wherein multiple compositions, illustrated in FIG. 1, are connected via a polypeptide linker. The first subunit, is a major or minor (e,g.
ETEC class 5 adhesin) ETEC fimbrial subunit. One or more major ETEC fimbrial subunits are then connected to the first subunit and to each other via a linker, wherein the subunits are stabilized by donor strand complementation. The C-terminal most ETEC

5.

major subunit is connected, via a linker, to a donor strand region from an ETEC major subunit, which can be either homologous or heterologous to the temnnal major subunit.
In some construct examples, in order to avoid inadvertent association of subunits, especially in CS6 subunits to each other, major ETEC fimbrial subunits can contain an N-terminal deletion of 14 to 18 amino acids.
'FIG. 3. SDS-PAGE and immunoblots of conjugate vaccines. A. Analyses of CfaE-1-1536 conjugate. Lane 1-3 are stained with Gel Code Blue. Lane 1, Precision Plus Protein standards (BioRad); lane 2, CfaE; lane 3, CfaE-HS36 conjugate. Lanes 4-5 are immunodetected with anti-CfaE antibodies. Lane 4, CfaE; lane 5. CfaE-HS36 conjugate.
Lanes 6-7 are immunodetected with antibodies to whole cells of 81-176 (HS36).
Lane 6, CfaE-HS36 conjugate; lane 7, proteinase K. digested whole cells of 81-176. B.
Analyses of CfaEB-HS36 conjugate. Lane 1-3 are stained with Gel Code Blue. Lane 1, Precision Plus Protein standards (BioRad); lane 2, CfaEB lane 3, CfaEB-HS36 conjugate.
Lanes 4-are immunodetected with anti-CfaE antibodies. Lane 4, CfaEB; lane 5, CfaEB-conjugate. Lanes 6-7 are immunodetected with antibodies to whole cells of 81-(11S36). Lane 6, CfaEB-HS36 conjugate; lane 7, proteinase K. digested whole cells of 81-176. The molecular weights of the protein markers are shown on the left.
FIG 4. C. Aittni anti-CPS (A) or ETEC anti-CfaE (B) induced hy HS36 conjugated to CfaE or CfaEB in mice.
FIG. 5. Functional antibodies, evidenced by HAI titer, induced in mice immunized with 1-1536 conjugate vaccines.
FIG. 6. Summary of synthesis of polysaccharide construct and conjugation to CR1v1197,

6 FIG. 7. Synthesis of aminopentanyl OMe-phosphoramidate galactoside. Reagent and conditions: (a) TrCI, pyridine, 95%; (b) AllBr, NaH, IF, 0 C, 89%; (c) CAN, CH3CN, 1-120, 0 C; then CCI3CN, K2CO3, CH2C12, 57% over 2 steps; (d) HO(CH2)5NPhth, TMSOTf, CH7C12, 65%; (e) 80% AcOH, 80 78%;
(f) PCI2024e2, Et3N, CHC1, then NH3(g), 27%; (g) PdC.12, Me01-1, 75%, (h) H2NNI12, Et0H, 82%.
FIG. 8. Capsule cross-reactivity to 6-MeOPN-Ga1 with antibodies to multiple conjugate immunogenic compositions.
FIG. 9. Serology of A. nancyrnaae immunized with CfaEB-HS23-36 construct. Day verses day 140.
FIG. 10, HA I titers of A. nanaeyinaae against ETEC strain1110407 expressing Cfa I.
FIG. 11. Immune response of mice against HS3 capsule (top panel) and against (bottom panel) following immunization with an CssBA-HS3 conjugate vaccine. The vaccine was administered at two doses, either 5 jig or 25 jig by weight.
FIG. 12. Immune response of mice to HS 4 capsule (top panel) and to LTB
(bottom panel) following imniunization with an LTB-HS4 conjugate vaccine. The vaccine was administered at two doses, 5 ug or 25 ug by weight.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0013] The term enterobacteria , as used herein, refers to enterotoxigenic Escherichia coil (ETEC), Campylobacterjejuni or Shigella spp., %which include: Shigella dvsenteriae, Shigeliallexneri, Shigeila Boydii or Shigella sonnei, As used herein, an enterobacteria polysaccharide polymer is a polysaccharide polymer derived from enterobacteria. The

7 tern "polysaccharide antigen" as used herein refers to a capsule polysacchride derived from Campylobacterjejani (G. jejuni or C'ampylobacterjejuni capsule) or a lipopolysaccharide derived from Shigella spp.. As used herein, "polysaccharide" refers to two or more monosaccharide units composing a carbohydrate polymer molecule.
A
"polysaccharide polymer" refers to two or more polysaccharide molecules connected together.
[0014] The terms "polypeptide," "peptide," and "protein" as used herein can be interchangeably used, and refer to a polymer formed of two or more amino acid residues, wherein one or more amino acid residues are naturally occurring amino acids.
The term "amino acid sequence" refers to the order of the amino acids within a polypeptide. As used, herein, "oligomer" are polypeptides sequences comprising relatively few amino acids.
[0015] The term "recombinant potypeptide", "remnbinant polypeptide construct", or "recombinant protein", as used herein, refers to polypeptides or proteins produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or the desired protein. The term "recombinant construct" refers to the DNA encoding the recombinant polypeptide, recombinant polypeptide construct or recombinant protein.
[0016] The tern "donor strand" or "donor strand" refers to the N-tenninal region of an ETEC fimbrial subunit that associates with another ETEC fimbrial subunit in donor strand complementation.
[0017] The term "immunogenic composition" refers to a formulation containing proteins or polypeptides or polysaccharides or polysaccharide polymers that induce a humoral

8 and/or cellular immune response. The term "immunogenic coverage" or "spectrum of coverage" refers to the induction of Immoral and/or cellular immune response against specific strains of bacteria under the "coverage." The term "immunogenic fragment"
refers to a polypeptide containing one or more B- or T-cell epitopes and is of sufficient length to induce an immune response or to be recognized by T- or B-cells. The term "derivative" refers to a pol5peptide or nucleic acid sequence with at least 80% identity with sequence of the identified gene. In this context, "identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when aligned for maximum correspondence. Where some sequences differ in conservative substitutions, i.e., substitution of residues with identical properties, the the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Percent similarity refers to proportion of identical and similar (conserved change) residues.
[00181 "Fimbriae" are defined as projections or filaments on ETEC bacteria arid are composed of major subunits, as in the case of CS3 and CS 6 fimbriae or major and minor subunits, as in the case of class 5a, 5b and 5c ETEC, "Fibrillae" are narrow projections from a bacteria. CS3 and CS 6 fimbriae can also be termed fibrillae due to their narrow characteristic. The term "fimbrial subunit" refers to the proteins that comprise ETEC
firnbriae and is used interchangeably with "pilin." "Pilin", therefore, can refer to a "major" or "minor" "fimbrial subunit" that comprise ETEC fimbriae. .A "minor fimbrial subunit" refers to the adhesin protein at the tip of class 5 ETEC firnbriae and is expressed in stoichiometrically low amounts compared to "major" subunits. The "minor firnbrial subunits"include, but are riot limited to, CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD

9

10 and Cota "Major fimbrial subunits" refers to the ETEC fimbrial proteins represented in stoichiometrially larger amounts in ETEC firnbriae, compared to "minor fimbrial subunits." "Major fimbrial subunits" include the ETEC class 5 proteins: CfaB, CsfA, CsuA2, CsuAl, CooA, CosA, CsdA, CsbA, CotA; the ETEC C53 proteins: Cstli, CstG;
and the ETEC CS 6 proteins: CssA, and CssB, [00191 The pathogenesis of Cl:ampylobacterjejuni remains poorly understood in comparison with ETEC and the organism shares few virulence factors with better-characterized pathogens. C. jejuni is unusual, however, among enteric pathogens in that it expresses a polysaccharide capsule (CPS) that is one of its few confirmed virulence factors.
[0020] Because of the importance of ETEC and C jejuni as pathogenic agents, a combined ETEC-CJ composition was constructed in order to afford protection against both agents. In one embodiment, a recombinant polypeptide construct, comprising fimbrial subunits from Class 5 ETEC strains is fused to a capsule polysaccharided froin the C jejuni strain 18-1'76.
[0021] in a preferred embodiment, one or more recombinant polypeptide ETEC
constructs, comprising the ETEC fimbrial adhesion, are conjugated to isolated C. jejuni capsule polysaccharide (CPS). One or more of a number of ETEC recombinant constructs can be conjugated to one or rnore of a number of C. jejuni capsule polysaccharide structures. In the inventive construct, the ETEC recombinant construct operates both as an inununogen against ETEC and as a protein carrier molecule, presenting the C jejuni polysaccharide. Examples of ETEC recombinant polypeptides and C. jejuni capsule polysaccharides that can be incorporated into a combined structure are given in the following examples.
[0022] In a preferred embodiment, the ETEC polypeptide construct can not only serve as antigen against ETEC but also serve as a protein carrier for polysaccharide antigens, such as C. jejuni capsule polysaccharide.
.Example 1: Conjugation of ETEC polypeptides to C. jejuni capsule polysaccharide [0023] In a preferred embodiment, ETEC recombinant polypeptides or polypeptide constructs are conjugated to C. jejuni CPS, The CPS can be derived from a number of C.
jejuni strains. In the embodiment, any CPS of any C. jejuni strain is envisioned to be conjugated to ETEC recombinant polypeptide constructs. Alternatively, Shigella LPS
can be conjugated to ETEC recombinant polypeptide constructs, [0024] The overall method of conjugating includes oxidizing C. jejuni CPS, for example, with NaI04 in sodium acetate (pH 4,0). Oxidized CPSs were desalted with a 5 Ic.Da cutoff membrane by stirred ultrafiltration, which is subsequently lypholized.
ETEC
proteins are then added. The stoichiornetery protein to CPS can vary, however, a typical ratio is 1:2 protein to CPS by mass. The concentration of components can be by any method. However, for exarnple, polysaccharide concentration was determined by antrhone assay and protein concentration was detemiined. NaCNBH3 is then added. The conjugates are subsequently desalted by ultrafiltration and lyophilized. CPS
(or Shigella LPS), ETEC proteins and conjugates were analyzed, for example by SEC-HPLC.
Conjugates were also analyzed by SDS polyacrylaminde gel electrophoresis (PAGE) and Gel Code Blue (Pierce, Biotechnology, Inc, Lombard, IL) staining. Conjugates were detected by antibody-based assay using anti-CPS and anti-ETEC protein.
[0025] As an example of ETEC recombinant polypeptide and C. jejuni conjugation, the CPS from the C jejuni strain 81-176 was conjugated to ETEC recombinant polypeptide construct CfaE (class 5 ETEC adhesin) or to the recombinant polypeptide construct CfaE
linked, via a polypeptide linker, to the major subunit CfaB.
[0026] CPS C jejuni capsule was purified from Campytobacterjejuni strain 81-(PG3208). This mutant, in which the gatT gene was insertionally inactivated by chloraphenicol cassette, lacks all ganglioside mimicry in its lipoligosaccharide (LOS) core [0027] The cells were grown in porcine Brain-Heart Infusion (BHI) broth and sonicated to inactivate the cells. The CPS was extracted by hot water/phenol method previously employed for the same organism (Chen, et al., Carbohyd. Res. 243: 1.034 (2008)). Cells were immersed in a water/phenol mixture (3:2 ratio by volume), which was heated to 67.c with stiffing for 4 hours. The suspension was cooled and separation of the mixture into two separate layers (the aqueous layer a.nd the phenol layer) and extraction of the aqueous layer was performed. The aqueous layer was removed and the phenoliwater extraction was repeated on residue, to maximize the yield. Aqueous 'layers from two extractions were pooled arid boiled fro 1.5 hours with the aditon of acetic acid to a pH of 3.5. The aqueous layer was dialyzed gairist running water for 2 days and concentrated using a Millipore concentrator cell with a 5 kDa cutoff membrane. Trace amounts of residual RNA were removed by digestion with benzonase enzyme at 90 tilmi in 50 mM
Tris-CC1/1 mM MgC12, pH 8 overnight at 37 C. Benzonase was removed from CPS, then desalted and concentrated using stirred ultrafiltration with 30 and 5 kDa MWCO
disc membranes, respectively.
[0028] The isolated CPS was oxidized with adding 40 mg of CPS to 40 mNi NaI04 in sodium acetate pH 4 in the dark at 4 C for 2 days. Oxidized CPS was desalted with 5kDa cutoff membrane by stirred ultrafiltration and was subsequently lyophilized.
[0029] Prior to conjugation the ETEC proteins, for example d.scCfaE and dscCfaEB, were transferred to 0.1M borate buffer at pH 9Ø Oxidized CPS was added to each ETEC protein at a ratio of 1:2, protein to CPS by mass, and then NaCNBH3was added at 2 times mass equivalent to CPS. The reaction was incubated 1 day at room temperature and 6 days at 37 C in the dark with continuous stirring. The conjugates were desalted by stirred ultirafiltration with 30 k-Da membrane and lyophilized. Conjugates of the CPS to dscCfaE and dscCfaEB was conducted by SEC-HPLC, and polyacrylamide gel (PAGE) (12.5%) electrophoresis.
[0030] In PAGE analysis, immunodetected with rabbit polyclonal antibodies to whole cells of 81-176 was used to detect CPS and to CfaE. The results of this study are shown in FIG. 3. Immunoblotting of both conjugates with anti-CfitE antisera confirmed that the proteins ran as high molecular weight conjugates with conjugates with apparent masses ranging from just higher than the mass of each respective protein to >250 kDa.

Immunoblotting with antisera to formalin fixed whole cells of C. jejuni 81-176 confimis that capsular polysaccharide was conjugated to the proteins. As illustrated in FIG. 3, no unconjugated protein remained in either conjugation.
[0031] The results of FIG. 3 were confimied in SEC-HPLC, hi the SEC-HPLC, unoxidized and oxidized CPSs, ETEC proteins and conjugates were analyzed using SEC-O

FIPLC with a TSKgel-G2000SW,dcolumn (30 cm x 7.8 mm ID) and TSKgel SW guard column run on an ICS-5000 Dionex system with 0.1 1\4 phosphate at pH 6.8, 0.1 M
sodium sulfate and 5% acetonitrile at 0.6 ml/min flow rate. Samples were monitored at 214 rim with Ultimate 3000 variable wavelength detector and RI detector, both from Dionex.
[0032] The results of the SEC-HP LC are shown in FIG. 4 for dscCfaEB and in FIG. 5 for dscCfaE. Analysis by matrix-assisted laser desorption/ionization (MALDI) is shown in FIG. 6, for dscCfaEB and FIG. 7 for CfaE.
[0033] Detection of the conjugates by refractive index (RI) on SEC-HPLC
revealed that 45% and 50% of the polysaccharide remained unconjugated with the CfaE and CfaEB
conjugates respectively. This is summarized in Table I, which also illustrates that the conjugated molar ratio of CPS to CfaE was 4.8:1 and that of CPS to CfaEB was 4.4:1.
Table 1 CfaE conjugate CfaEB conjugate CPS CfaE CPS CfaEB
Final product (includes unconjugated CPS) % yield 49% 63%
Sugar : protein mass ratio in final product 2 1 L85 1 % unconjugated with respect to final product by SEC-HPLC RI detection 45% 50%
Conjugated mass ratio 1 1.5 1 2.3 Molecular weight 5.5 kDa 41 kDa 5.5 kDa 57 kDa Conjugated molar ratio 4.8 1 4.4 1 Example 2: Anti-Class 5 ElEc CS3 or CS' constructs [0034] Anti-ETEC constructs that are contemplated to be conjugated to C j4uni polysaccharide comprise the structures as illustrated in FIG. 1 and FIG. 2.
FIG, 1 illustrates the basic recombinant construct design. As diagrammed in FIG. 1 the construct design comprises one, or more ETEC major or minor fimbrial subunits or fragnents of major fimbrial subunits, containing the donor strand, derived .from the same ETEC fimbrial type, which are connected, via polypeptide linkers and stabilized by donor strand complementation. The construct can contain a deletion of the N-terminal region of the I'1-terminal subunit. This feature prevents undesirable associations with other monomers or multimers, The C-tenninal subunit is connected to and stabilized by a donor 13 strand, connected to the subunit via a polypeptide linker, wherein the donor p strand is either derived from the adjacent subunit (i.e., homologous) or from a different subunit of the same fimbrial type (i.e., heterologous).
[0035] FIG, 2 illustrates the. basic multipartite construct, wherein multiple constructs as in FIG. I, are connected forming a recombinant construct comprising two or more firnbrial typesõAs illustrated in FIG. I, major or minor subunits from the same fimbrial type are connected via a polypeptide linker sequence. In the multipartite construct, two or more constructs, as in FIG. I. are connected, via a linker polypeptide, [0036] In the multipartitie construct design, as in the basic design (compare FIG. I with FIG, 2), the first subunit (N-terminal) is a major or minor ETEC fimbrial subunit Each additional subunit is connected to adjacent subunits via a polypeptide linker that enables rotary freedom of the molecular components. The subunits are associated with and stabilized via a donor strand complementation from a C-terminally adjacent subunit via a donor 13 strand, connected via a linker polypeptide, to the C-terminus of the stabilized subunit. In some embodiments, subunits can contain a deletion of 14 to 18 amino acids from its N-terminal end. Additionally, specific constructs can be constructed with or without signal peptides of 18 to 22 amino acids and with or without histidine tags at the C-terminus.
[0037] In the multipartite construct, subunits 'from the same fimbrial type are directly connected. Groupings of subunits from the same fimbrial type are then connected to other groupings of subunits from other fimbrial types. Fimbrial types include, but are not limited to ETEC class 5a, 5b, 5c, CS3 and CS6. For example a single construct can include subunits derived from any two or more of class 5a, 5b, 5c, CS3 and CS6 fimbrial types.
[0038] Multiple linker sequences can be utilized in connecting the individual subunits.
Examples of specific linkers include the tetrapeptide of SEQ ID No. 5. Another example is a tri-glycine linker (i.e., G-G-G). In the inventive construct, in cis donor strand complementation is used to stabilize adhesins and adhesin-pilin fusions for representative Class 5a, 5b, and 5c adhesins.
[0039] The contemplated composition is designed to enable as wide a range of coverage of ETEC strains as possible. As such, in one embodiment, the contemplated composition and use is aimed at inducing immunogenic response against class 5a, 5b, 5c ETEC, as well as ETEC strains expressing CS3 or CS6 fimbrial components, [0040] In a preferred embodiment, recombinant polypeptide ETEC constructs are conjugated to C jejuni capsule polysaccharide (CPS). One or more of a number of ETEC recombinant constructs can be conjugated to one or more of a number of C.
jejuni capsule polysaccharide structures. Examples of Class 5 ETEC recombinant polypeptides are listed in Table 2. In Table 2, minor subunits are stabilized by connection, via a polypeptide linker, to associated major subunits. Alternatively, a 12-16 amino acid donor strand, derived from the associated major subunit is connected to the minor subunit via a polypeptide linker. These polypeptides can also be linked as per FIG. 1 and FIG. 2 to lead to the example constructs listed in Table 3. These examples can then be conjugated to isolated C jejuni capsule polysaccharide, as in Example L
Table 2 i SEQ ID No. _____________________ 1 hnmune SEQ 1D No.
coverage Subunit 1 Fulllength sequences Mature sequences inult3cling spdi (fimbrial types) (DNAlpolypeptide)2 (DNAipolypeptideD
....................... -,, Class 5a CfaE 1 ______ 56/57 115/58 --________________ CfaB 59/60 ............................ 116/61 CstD 64/65 117/88 --CsfA 62/63 ............................................... 118/89 CsuD 70/71 119/90 --Csu A2 68/69 120/91 CsuAl . 66/67 ................ 121/92 Class 51) ...... Coop 74/75 ----------------------------- 122/93 CooA 72/73 ................ 123/94 _ Csdn 78/79 124/95 CsdA 76/77 125/96 (.7s1 82/83 133/97 CosA 80/81 126/98 ¨
.................................. CsbD 44/45 127/46 CsbA 47/48 128/49 Class 5c Co1D 50/51 129/52 CotA 1. 53/54 130/55 CS3 ............ Cs11-1 84/85 131/99 ..
___________ 1 CstG /. 86/87 ---------------- 132/101 CS6 CssA = 134/135 1/2 i 1 CssB i 136/137 3/4 I "spd" refers to signal peptide. The mature polypeptide sequence, therefore, would be the full length minus the signal peptide.
2DNA sequence encodes mature protein.
1=1 Table 3 Fimbriae class SEQ ID No,, represented Construct (adhesin-pitin) examplel (ProteiniDNA)3 Class 5a cisci4c.FACfaE-CfaB-Csu...A2-CsfA 103/104 Class 5b dsci4csbACsbD-CsbA-ntdi5dscl4cooACooA2 105/106 Class 5b dsci5ACsbD-CsbA-CooA2 107/108 Class 5c = dsel4e0tACetD-CotA 1091110 Idsc refers to donor strand complementation. The number and subunit refers to the 1'J-terminal amino acids of length represented by the number from the subunit indicated that is connected at the C-terminus of the construct and is serving to stabilize the C-terminal construct. For example, "dsci4c.8rA" refers to the N-tenninal 14 amino acids of CsfA. connect to the C-terrninus of the construct.
Linkers polypeptides are GGG rather than DNKQ.
3Sequence in example contains a Leu-Glu-His6 at the C-terminus.
[00411 An important feature of the anti-ETEC construct is the enhanced iMMUirie recopition of the fimbrial adhesion. The minor subunits (i.e.. ETEC adhesin) of ETEC
Class 5 firnbriae are stoichiometrically represented in very low numbers relative to the major subunit. Therefore, an important feature of the recombinant constructs is the vastly improved stoichiometric representation of the minor subunit in order to enhance immune recognition of the minor subunit. Additionally, since 'firnbrial subunits, such as CfaE, are relatively susceptible to proteolytic degradation outside of the firnbrial structure, stabilization of the adhesin is also important. Therefore, constructs are designed to express ETEC subunits stabilized from misfolding and degradation by donor strand complementation.

[0042/ The donor [3 strand is provided by the major fimbrial subunit. For example, in the case of CfaE, stabilization is provided by the N-terminal region of CfaB.
Engineering of dscCfaE by incorporation of a donor peptide strand from the N-terminus of the CFA/I
major subunit CfaB at its C-terminus transformed an insoluble, unwieldy native, recombinant protein into a stable immunogenic composition (Savarino, U.S.
:Patent application publication no. 20060153878 (13 July 2006)), which is incorporated by reference, herein.
[00431 Based on its atomic structure, dscCfaE is folded into a native, 13-sandwich conformation, consisting of two half-barrels, comprising the N-terminal adhesin domain (CfaEad) a short a-helical connector, and the C-tenninal pilin domain (CfaEpd). The molecule is functional in that it directly mediates MR-IA of bovine and human erythrocytes, and generates neutralizing antibodies that act to inhibit MRIL1A
and decorate the tips of CFA/ firnbriae on immunoelectron microscopy.
[0044] A fusion protein was engineered by genetic insertion of the coding sequence for mature major structural subunit of ETEC adhesin, such as CfaB, to the 3'-end of the minor subunit, such as CfaE. This concept was disclosed in Savarino, U.S.
Patent application (111340,003, filed January 10, 2006), which is incorporated, herein, This molecule contains all three domains of the CFA/1 fimbriae (i.e,, ad, pd, and major subunit) in a ratio of 1:1:1, rather than that found in native fimbriae (ca.
1:1:1000).
[0045] A number of observations indicate the suitability of dscCfaE (cloned from ETEC
strain E7473) as a vaccine antigen. First, sequencing of 31 different wild type alleles of cjiiE from ETEC isolates of varying geographic origin and serotypes, show that the gene and predicted polypeptide sequence are nearly invariant, with three different nonsynonynous nucleotide changes at one site each in only five of these 31 alleles (Chattopadhyay, et al., Biol. Chem., 287(9): 6150-6158 (2012)). Hence, the target protein shows uniformity in natural ETEC bacterial populations.
[0046] Additionally, CfaE, a Class 5a fimbrial adhesin, is 80-81% identical with the other Class 5a minor subunits proteins adhesins CsuD of CS14 tirnbriae and CstD of C54 fimbriae, CsuD and CsiD share 94% identity. This is considerably higher than the average identity with other Class 5b and 5c fimbrial adhesins (mean 50%
identity).
[0047] Moreover, rabbit anti-dscCfaE serum cross-neutralizes CS4- and CS14-ETEC in the hemagglutination assay (HAI). A number of vaccination studies have been performed in small (rabbit and mice) and large (monkeys and cows) animals with various routes of administration and adjuvant combinations showing that dscCfaE is a potent immunogen that can elicit systemic and mucosal antibodies which recognize dscCfaE and CFAII and are neutralizing (as measured by HAI assay), [0048] An embodiment includes anti-class 5 ETEC constructs based on the construct design illustrated in FIG. 1, whereby the N-terminal subunit is an ETEC class 5 minor (i.e., adhesin) subunit, listed in Table 2, including CfaE, CsfD, CsuD, CooD, CsdD, CosD, CsbD and CotD, connected, via a polypeptide linker, to one or more ETEC
major subunits, from the same ETEC class 5 type, listed in Table 2. The polypeptide linker can be any of a number of polypeptide sizes. in a preferred embodiment, the link-er is a tetrapeptide with the polypeptide sequence of SEQ ID No. 5. The C-terminal class 5 subunit is connected to a donor 13 strand, derived from a homologous subunit and is typically 12-19 amino acids. In alternative embodiments, one or more major subunit can include a deletion of 12 to 16 amino acids from the N-tem-iinal region of the subunit.

[0049] The design in FIG. I, utilizes the concepts disclosed in Savarino, U.S.
Patent application (11/340,003, filed January 1), 2006)), including don.or strand com.plementation to provide stabilized class 5 ETEC adhesin. Due to the homology of ETEC class 5 minor subunits and major subunits, FIG, 1 further contemplates multiple constructs incorporating the fimbrial subunits of Table 2, or derivatives of these polypeptides or DNA sequences.
[0050] The construct design, illustrated in FIG. 1, incorporates the donor strand complementation stabilization features of Savarino (U.S. Patent application (11/340,003, filed January 10, 2006)), and furthers it by incorporating multiple major subunits, 'from a specific ETEC type, into a single adhesin-pilin construct. For example, multiple class Sh major subunits can be connected to a class 5b adhesin (i.e, minor subunit), Embodiments include adhesin-pilin constructs containing Csb D (ETEC Class 5b fimbrial adhesin) and Cot D (ETEC Class 5c firnbrial adhesin). Examples, for illustration, of embodiments of adhesin-pilin ETEC class 5 adhesin-pilin constructs, representing Class 5a, 5h and 5e are shown in Table 3, CS6 and CS3 [0051] Rabbit model (RITA.RD) studies suggest the colonization factor CS 6 and CS3 has immune-protective potential (Svennerholm, et al., Infect. Immun. 56: 523-528 (1988);
Svennerholm, et al., Infect. Immun, 58: 341-346 (1990)). As such, an important technical goal is to reproduce a stabilized CS 6 expressing recombinant structure expressing CS 6 antigens that maximally elicits antibody responses inhibitory to CS6-directed adhesion.

[0052] Unlike class 5 ETEC fimbriae, the fimbrial structures rnay function as polyadhesins rather than monadhesins (Zavialov, et al., FEMS IVIicrobiol, Rev, 31: 478-514 (2(07)). Extrapolation from related fimbriae, assembly of ETEC CS(. and CS3 may be mediated by a donor strand complementation mediated process through association of a CS 6 or CS3 subunit with the N-terminal donor strand region of an adjacent subunit.
Additionally, protection against misfolding and proteolytic degradation may also be afforded through donor strand coinplementation.
[0053] Association of monomers of CS3 and CS 6 was evaluated by visualizatioa of the subunit proteins under denaturing and non-denaturing conditions in polyacrylamide gel electrophoresis (PAGE). For both CS3 and CS 6 monomers, under den.aturing conditions the proteins migrating at the expected sizes. Under non-denaturing conditions multiple size (i.e., ladders) are seen formed by multinieric association of the subunits.
C'S6 fimbriae [0054] CS 6 fimbriae comprise CssA and CssB. Whereas the two CS3 major subunits show little to no variation ia polypeptide sequences, modest variation in CS( proteins is observed. For example, greater than 90% identity is found in CS 6 protein CssA
and greater than 95% identity is found in CssB allotypes. Both CS 6 structural proteins exhibit a relatively low level of variation (i.e., greather than 90% amino acid conservation), with greater variation in CssA and the mutations randomly distributed along the CssA polypeptide.
[0055] In order to design an effective immunogenic composition that would be suitable for inclusion in a vaccine formulation a number of criteria were devised for determination of suitable constructs. These included the ability to maintain a structure without unwanted self-association or assembly; thermostability; and ability to generate anti-CS6 IgG and IgA antibody levels similar to those elicited by immunization with CS.

[0056] Monomeric CS 6 subunit assembly appears to be mediated by donor strands from adjacent CS 6 subunits, as discussed above. It is hypothesized that interaction to form these stable structures is mediated by inter-subunit interaction through donor strand complementation. Donor strand complementation also affords protection against misfolding and proteolytic degradation. Therefore, in a preferred embodiment, multimeric CS 6 constructs were developed to take advantage of these attributes of donor strand complementation. Additionally, multimeric expression provides more efficient manufacture over production of monomers, [0057] In one embodiment, a construct conjugated to C. jejuni comprises a multimeric CS61,vith one or rnore of the CS 6 subunits, CssA and CssB, or allelic variation or derivatives, with the construct design configuration illustrated in FIG. 1. In a preferred embodiment, the construct comprises a dirner of CssB and CssA with Css13 N-terminal to CssA (i.e., CssB-CssA), [0058] As illustrated in FIG, 1, or FIG, 2, CS 6 subunit ;association is stabilized by in cis donor strand complementation. Donor strand complementation is afforded by linking a CS 6 subunit at its C-terminus, to the. donor i3 strand region of another CS6 subunit, via a tetrapeptide linker. The linker can be any of a number of polypeptide regions.
However, in a preferred embodiment, the linker is either as in SEQ ID No, 5 or a triglyicine linker.
In the case of a terminal CS 6 subunit, stabilization is provided by donor 3 strand, connected at its C-terminus, from a homologous or heterologous CS 6 subunit, Homologous subunit is defined as two subunits of the same form (e.g., CssA OR
CssB).
Heteterologous subunits are of different forms (e.g., one is derived from CssA
the other from CssB. The CS 6 donor p strand is typically the N-terminal 14-16 amino acid region of CS 6 subunit. The recombinant protein can be constructed with or without hexahistidine affinity tags, which are typically on the C-terminus.
[0059] Additionally, to prevent recombinant polypeptide constructs fuming molecular associations resulting in un-desirable non-covalent oligomer forniation, in a preferred embodiment, the N-terminal 14-16 amino acids of the N-terminal CS 6 subunit is deleted.
As an illustration, "dscB14CssBA" would contain a heterologous donor strand (i.e., "dsc"), from CS 6 CssB, inserted at the C-temiinus of the construct. In this case, the donor strand is 14 amino acids in length, as indicated by the "14." Similarly, a constructed designated "ntdisdscACssBA" would contain a homologous donor strand at the C-terminus of the construct and also comprises a deletion of the N-terminal amino acid region (termed "ntd").
[0060] Examples of constructs comprise one or more CS 6 subunits with earning acid sequences sequences selected from the group consisting of SEQ ID No. 2 (CssA) or SEQ
ID No. 4 (CssB), or derivatives of these polypeptides. The DNA sequence for CssA is SEQ ID No. I and for CssB, SEQ ID No. 3. The subunits are connected by a polypeptide linker sequences. In a preferred embodiment, the linker is a tetrapeptide with the amino acid sequence of SEQ ID No. 5.

CS3jimbriae [0061] Savarino (U.S. Patent application No. 11/340,003 (2006)) discloses donor strand complementation stabilized ETEC constructs, Embodiments of this application incorporate the donor strand stabilization of CstH and adds the second CS3 subunit, CstG. Embodiments herein add additional features found to be important for stabilization of the CS3 subunits and immunogenicity against CS3. CS3 comprises CstH and CstG.
The CS3 structural protein CstH is invariant. CstG is also highly conserved, showing 99-100% identity in poly-peptide sequence for 39 wildtype CS3 genes sequenced.
Similiarly, although some variation CstG is observed, it is also relatively invariant, with 99-1)0%
amino acid conservation.
[0062] CS3 contains both CstG and CstH, in near equal amounts. Therefore, dirneric constructs were devised incorporating CstG and CstH, according to the template construct design of FIG. 1.
[0063] in one embodiment a polypeptide construct conjugated to C. jejuni capsule polysaccharide comprises a CS3 s construct designed according to FIG. I. In FIG, I, CS3 constructs comprise one or more CS3 fimbrial subunits connected via a polypeptide linker. The C-terminal fimbrial subunit is connected, via a polypeptide linker, to a donor fi strand region of a CS3 fimbrial subunit. The C-temainal donor p strand can be derived from the same CS3 subunit to which it is connect (i.e., homologous) or derived from a different subunit (i.e., heterOlogous). The polypeptide linker can be any number of polypeptide regions, however, in a preferred embodiment, the linker is a tetrapeptide of the sequence of SEQ ID No. 5, or a triglycine (i.e., G-G-G). The donor p strand region is the N-terminal 14 - 16 amino acids of the mature CstH or CstG protein. In alternatives of this embodiment, the first 14 - 18 amino acids of the N-terminal region of the N-terminal most subunit is deleted to avoid undesirable associations.
[0064] In a preferred embodiment, the CS3 construct is a dimer. Although other exarnples are contemplated using the design of FIG. 1, as an illustrative example, the recombinant polypeptide construct can be configured as "dsci6csalCstG-(1inker)-CstH".
In this example, the mature CstG polypeptide (SEQ ID No. 101) or fall length polypeptide sequence (SEQ ID No. 87) is connected at its C-tenninus to CstH
polypeptide (SEQ ID No. 99), via a polypeptide linker. In this example, the CstH
polypeptide, is connected, at its C-terminus, to a donor 0 strand region of 16 amino acids derived from CstH via a polypeptide linker.
[0065] Other examples can include constructs, according to FIG. 1. In other examples, the C-tenrninal donor p strand can be either homologous (derived from the same subunit) or heterologous (derived from a different subunit) to the C-terminal most CS3 fimbrial subunit.
Construction of multipartite fusion constructs [0066] Immunity to multiple strains of ETEC is important to obtain the greatest extent of anti-ETEC immunity. Toward this goal, recombinant polypeptide constructs were developed comprising two or more subunits derived from different ETEC fimbrial types according to the design illustrated in FIG. 2 to form multipartite fusion constructs. As used, herein, multipartite fusion or multipartite fusion constructs are recombinant polypeptide constructs according to FIG. 2. In this design, different ETEC
fimbrial types are defined as fimbrial proteins derived from fimbriae of different strain ETEC types, as listed in Table 4 or 5, or deriviates of these polypeptides or DNA sequences.
For example, the fimbrial type "CS3" comprises Cst1-1 and CstG. The fimbrial type "CS"
comprises CssA and Css.B. The fimbrial types of Class 5 ETEC include the fimbrial types Class 5a, Class 5b and Class 5c, [0067] In a preferred embodiment, major andlor minor subunits, derived from the same ETEC fimbrial type are connected, via polypeptide linkers, and stabilized by donor i3 strand complementation, as illustrated in FIG. 1. A multipartite fusion comprises one or more fimbrial subunits of the same fimbrial type, as in FIG. 1, connected to one or more fimbrial subunits derived from a different fimbrial type as illustrated in FIG. 2.
[0068] In one embodiment, the multipartite fusion construct can include a deletion of the N-terminal region of one or more fimbrial subunits, but is preferably on the N-terminal most fimbrial subunit for a given ETEC fimbrial type, as illustrated in FIG.
2. This feature prevents undesirable associations with other monomers or multimers.
The size of the deletion of the N-terrninal region is 14 to 18 amino acids. In other embodiments, multipartite fusion constructs comprising Class 5 adhesins do not contain a deletion of the N-terminal region, [0069] As illustrated in FIG. 2, the C-terminal subunit, for an ETEC fimbrial type, is connected to and stabilized by a donor strand, connected to the subunit via a polypeptide linker, wherein the donor í strand is either that derived from the adjacent subunit (i,e., homologous) or from a different subunit of the same fimbrial type (i.e,, heterologous), The size of the N-terminal. donor strand depends on the fimbrial type and subunit stabilized. In preferred embodiments, for class 5 fimbrial subunits, the donor 0 strand, derived from the N-terrninal region of the class 5 subunit stabilized, is 12 to 16 amino acids. For C53 and CS 6 subunits, the donor strand is 14 to 16 amino acids. As mentioned above, the construct ca.n contain a deletion of the N-terminal region of the N-terminal subunit. This feature prevents undesirable associations with other monomers or multimers. The size of the deletion of the N-terminal region is 14 to 18 amino acids.
[0070] As illustrated in FIG. 2 multiple constructs as in FIG. I are connected forming a recombinant polypeptide construct comprising two or more ETEC firnbrial types.
In this way, one or more major or minor subunits, derived from the same ETEC fimbrial type, are connected via polypeptide linkers and stabilized by donor strand complementation.
In another embodiment, one or more glycine residues separates different ETEC
fimbrial types, acting as a "swivel" means between the ETEC types. The glycine residue, due to its small, unbranched molecular characteristics, enables rotary freedom of the molecular components. Subunits derived from the same fimbrial type (as in FIG. 1) are connected by a polypeptide linker, with the subunits stabilized by donor strand complementation.
As shown in FIG. 2, the C-temiinal subunit of each ETEC fimbrial type is stabilized by a donor p strand that is homologous or heterologotis to the C-terminal subunit of that fimbrial type, [0071] In other embodiments, the construct can contain an N-terminal deletion at the N-terminus of the entire construct as well as an additional deletion, of 14 to 18 amino acids, at the N-terminus of the first "internal" subunit that is of a different firnbrial type, This is illustrated in FIG, 2. In the case of the deletion on the N-tenninus of the "internal"
subunit, the deletion serves to shorten the length between subunits, thus reducing the likelihood of misfolding and proteolytic cleavage, hi another embodiment, a donor13 strand, derived from a homologous or heterologous subunit, is inserted at the C-termirius of the C-terminal CS 6 or CS3 subunit. For class 5 fimbrial subunits, the donor i3 strand, derived from the N-terminal region of the class 5 subunit that is stabilized, is 12 to 16 amino acids. For example, in preferred embodiments, CfaB is stabilized by a 14 amino acid donor 0 strand; CsfA by a 14 amino acid donor p strand; C.7sbA by a 1.5 amino acid donor p strand, CooA by a 14 amino acid donor 3 strand and Cot.A by a 14 amino acid donor p strand. For CS3 and CS 6 subunits, the donor p strand is 14 to 16 amino acids, with preferred embodiments of CS3 fimbrial subunits (i.e., CstEl. or CstG) stabilized by a 16 amino acid donor 0 strand derived from CstH or CstG; and CS6 fimbrial subunits (i.e., CssA or CssB) stabilized with a 16 amino acid donor p strand derived from CssA
or CssB. However, other donor p strand lengths are envisioned.
[00721 The inventive compositions can utilize different linker sequences. In a preferred embodiment, the linker contains the amino acid sequence of SEQ ID No. S. In another embodiment, the linker is a =tri-glycine linker. In other embodiments, the C-temainal end of the construct contains a histidine tag for purification of the construct.
[0073] In the inventive construct, in cis donor strand complementation is used to stabilize adhesins and adhesin-pilin fusions for representative Class Sa, 5b, and Sc adhesins, For each adhesin target group, in a preferred embodiment, the compositions are constructed with the intent of eliciting anti-adhesive immune responses. Further towards this goal, Class 5 multipartite fusions comprising Class 5 adhesin minor subunits are typically construct such that the adhesin (i.e., minor fimbrial subunit) is located at the N-terminus of the constructed with the minor fimbrial subunit linked at its C-terminus to one or more major subunits, followed at the terminal end of the construct with the donor P-strand of the last major subunit, [00741 Other embodiments include constructs comprising Class 5a adhesin CfaE
tandemly linked at its C-temiinus to one or more of CfaB (CFA1I major subunit), CsuA2 (CSI 4 major subunit) and CsfA (CSI major subunit); Class 5b adhesin CsbD
tandemly linked at its C-terminus to one or more of CsbA (CS17 major subunit), which shares high identity to the CS 19 pilin subunit CsdA, and CooA (C S1 major subunit), which shares high identity to the PCF071 pilin subunit CosA; and Class Sc adhesin Cot D
tandemly linked at its C-terminus to CotA (CS2 major subunit).
[0075] Embodiments of ETEC multipartite =f-Usion constructs are illustrated in Table 4 and 5. this embodiment, constructs comprise any major or minor ETEC
fimbrial subunit from Table 2 in multiple combinations, connected by linker polypeptides and stabilized from proteoly-tic degradation by donor strand complementation utilizing the design illustrated in FIG. 2, Table 2 lists the ETEC fimbrial subunits (major and minor subunits) than can be used and incorporated into the multipartite fusion construct design of FIG. 2, which can then be conjugated to C jejuni capsule polysaccharide or Shigelia LPS. Any subunit, therefore, is combined with one or more other ETEC major subunits from any ETEC fimbrial phenotypic type, including Class 5a, 5b, 5c, CS3 and CS.
[00761 The recombinant polypeptide construct motif comprises a whole or immunogenic fragment of a minor or major ETEC fimbrial subunit connected at its C-terminal end to a linker. The linker is connected at its C-terminus to a whole major ETEC
fimbrial subunit or a polypeptide donor strand of an ETEC major structural subunit, derived from the same firnbrial type. The whole ETEC major subunit or donor strand polypeptide is then connected, via a linker at its C-terminal end, to one or more additional major structural fimbrial subunits, derived from the same fimbrial type, from Table 2, [0077] The strategy for selecting and developing specific genetic fusion constructs is guided, in part, by the phylogenetic and antigenic relatedness of subunits.
For example, constructs containing Class 5a, 5b and Sc pilin subunits are selected based on the relatedness of minor and major subunits within a particular ETEC fitribrial class (i.e., class 5a, 5b or 5c). As such, adhesin (i.e., minor fimbrial subunit) from a specific fimbrial type (e.g., Class 5a) are linked to Class 5a major subunits. Further selection of subunits is guided and based on epidemiological study analysis in order to achieve optimum immunogenic coverage of FTC strains. What C. jejuni capsule polysaccharide to conjugate is predicated primarily on epidemiological data suggesting pathogenicity of the strain providing the capsule polysaccharide. Although many C, jejuni strains exist, most are not pathogenic.
[0078] In the multipartite constructs listed in Table 4 arid 5 the linker polypeptide, depending on the example construct, can comprise a four (4) amino acid sequence (tetrapeptide) or a tri-glycine. Also, as illustrated in FIG. 2, the subunits are interconnected and stabilized by donor strand complementation, which is denoted by "dsc". In this nomenclature, the fimbrial subunit derivation is also indicated. For example, in the construct "dscl6estH CStG-CStH-(G)-ntd sdSC16cõACssA-CssB", the N-terminal CS3 subunit "CstG" is connected, via a linker, to the CS3 subunit "CstH", which is connected, via a linker, to a donor strand of 16 amino acids derived from "CstH."
Similarly, the N-terminal CS6 subunit "CssB" is connected, via a linker, as illustrated in FIG. 2, to a 16 amino acid donor strand derived from "CssA." In this example, donor strand complementation of the "CssB"
subunit is via a heterologous donor strand (i.e., derived from "CssA)."
[0079] In Table 4 and 5 the examples contain a "G" (i.e., glycirie) to provide a "swivel."
,Alsoõ in some examples, the N-temainal region of N-terminal CS6 subunit is deleted (delineated by "ntd") to avoid undesirable association with other CS 6 subunits, as described above. It should be noted that, in addition to the examples illustrated in Table 4 or 5 (or Table 3), other combinations of major and minor subunits are contemplated utilizing the construct design illustrated in FIG. 2 and the fimbrial subunits of Table 2.
In some sequences listed, a six (6) histidine (i.e.,. His6) tag is inserted.
The constructs can be designed to include the histidine (i.e., His6) tag or designed without this tag region.
Additionally, some sequences contain the signal peptide (designated "spd" in Table 2 and 3) region. Constructs can be constructed with or without this region, as well, which may be added to improve manufacturing efficiency of the multipartite fusion construct.

Table 4 Firnbriai type (SEQ ID No. Examples of CS3 containing constructs!' 2' 4' 5 DNAProtein) Class 50./CS3 0/7) dsci4Cb.B-Cfa.E-CfaB-(C3-3)-ntEll8dsci6c,,HCstG-Cstli.
Class 5a1CS3 dscmestACfaE-CfaB-CsdA2-CsfA-(G)-ntdisdscl6cstfiCstG-CstH
Class 51-3/CS3 (10111) dsc N.õ,bACsbD-CsbA-ritdi5dsc34c,,CooA-(G)-ntd18dSeg 6e2tH CstG-Cs{
Class 5c/C53 (12/13) dsci4CotD-CotA-(G)-ntd38 dsci6rsttiCstG-CstEl CS3/toxin fusion (36/37) dscCstG-Csal -aCTA2 LTB multimeric composition (38/3)) CS3ICS6 (1.4/15) dsci6estaCstG-CstH-(G)-ntdItidsci6cõ,,,,CssA-CssB
CS6ICS3 (34/35) ntd14dscl6essaCssB-CssA-(G)-ritdi8dscu;cstuCstG-Cstil All combinations can include a histidine (i.e.. His) at the C-terminal end.
2 Subunits can be linked via either DNKQor tri-glycine linker.
3 (G) refers to glyeine residue introduced to provide a "swivel."
4 "ntd" refers to N-terrninal deletion (excised from mature protein) with extent of deletion. (i.e, amino acids) indicated.
"d,sc" refers to span of N-terminal residues from donor [3-strand, its amino acid length and its source.

Table 5 Fimbrial type (SEQ ID No. Examples of CS6 containing constructs DNA/Proiein) ind 4dSC 6cs,BCssB-CssA-(G)-ntdi8dsc eicstiiCstG-Cstll (34/35) CS3/CS6 dseloCsiCi-Cst11-(G)-ntd1.4ciseiKõBCssB-CssA
(32/33) Class 5b/CS6 spdt9dsci4cotACotD-CotA-(G)-indi4dscl6cõErCssB-CssA
(28/29) Class 5b/CS6 dsei4cwACotD-CotA-(G)-ntdi4dsci6c5sa-CssB-CssA
(30/31) C1ass5b/CS6 spd 9dse 5esbACsbD-(GGG)-CsbA4GGG)-ritdmdsci4c0DACooA-(G)-(GGG)-(24/25) ntdmdsoi6c8BCss13-CssA
Class 5b/CS6 dsetscRbACSIoD-(GGG)-CsbA-(GGG)-ntdmdsci4c.c.ACooA-(G)-(GGG)-(26/27) ntdi4dsei6c5saCssB-CssA
Class 5a/C56 dsci4ccu.sCfaE-CfaB-(G)-ntd6dsel6cssACssB-CssA
(16/17) Class 5a/CS6 rise 14c ihnCfaE-C faB-(G)-ntd i6dse i6cssliCssB-CssA
(11.3/114) Class 5a/CS6 dscl4ccasCfaE-Cfa13-(G)-ntd16dsc6essCssA-CssB
(18/1.9) Class 5a/CS6 dsel4craaCfaE-CfaB-(G)-ntd,;6dsel6csACssA-CssB
(11.1/112) dscl6csACssA-CssB-(G)-ritdi8dseleicgirstG-Csill (101/102) Class 5a/CS6 dsep,csfACfaE-CfaB-Csu.A.2-CsfA-(G)-ntdi4dseCssB-CssA
(22/23) Class 5a/CS6 (20/21) spd," dsewscACfaE-CfaB-CsuA2-CsfA-(G)-ntdi4dscCssB-CssA
CS6-chimera.
ntdi4dse I 6CssBCssB-CssA-sCTA2 (40/41) CS6-chimera nttl35dsci&cCssA-CssB-sCTA2 (42/43) I All combinations can include a histidine (i.e., is6) at the C-terminal end.
2 Subunits can be linked via either DNKQ or tri-glycine (GGG) linker. In preferred embodiments, DNKQ is used, except where indicated with (GC).
3 (G) refers to glyeine residue introduced to provide a "swivel,"
4 "spd" refers signal peptide. 'Number indicates number of amino acids.
"ntd" refers to N-terminal deletion (excised from mature protein) with extent of deletion (i.e., amino acids) indicated.
"dsc" refers to span of N-terminal residues from donor 0-strand, its amino acid length and its source, [00801 In another embodiment, recombinant polypeptide constructs can contain a C-terminal toxin A subunit, such as cholera toxin A2 (CT'A) to form a chimeric molecule.
In this embodiment, a full-length or truncated CTA2 is connected to CS 6 or multimeric recombinant polypeptide construct, such as a CS 6 or CS3 diner.
[00811 Examples of these toxin constructs are illustrated in Table 4 arid 5.
In these constructs, the LTB gene and the CS3 or CS 6 ¨ toxin chimera are separately expressed.
LTB, once expessed, would self assemble to form a pentarneric structure. The ensuing LTB multimeric composition (i.e., LTB5) and CS3 or CS 6 ¨toxin chimera then non-covalently associate to form a holotoxin-like heterohexarner.
[00821 Although other examples are contemplated, the sequences of examples of illustrative chimeric constmets, containing a C-terminal toxin component, are illustrated in Table 4 (for CS3) and Table 5 (for CS6).
[00831 For C83-chimeric molecules, one or more CS3 fimbrial subunits are connected, as in FIG. 1, via a polypeptide linker, preferably a tetrapeptide or triglycine.
The C-terminal most CS3 fimbrial subunit is then connected to a donor p strand, via a polypeptide linker.
The donor strand can be homologous or heterologous to the C-terininal fimbrial subunit.
The donor strand is then connected to a toxin fragment, such as CTA2, The CS3-chimera example shown in Table 4, comprise the polypeptide sequence of SEQ ID No. 37, which is encoded by the DNA sequence of SEQ ID No. 36. :In this example, the N-terminal fimbrial subunit is CstG with a pelB leader (22 amino acids) connected at its N-terminal end (see FIG. 13). However, different ordering of CS3 fimbrial subunit units is contemplated. Also, in this example, the CstH is connected, via a polypeptide linker, to a 16 amino acid donor strand derived from the N-terminal 16 amino acids of CstH, which is connected to an A2 toxin fragnent (i.e,, CTA2). In a preferred embodiment, LTB is also expressed. LTB comprises the amino acid sequence of SEQ ID No. 39 and is encoded by the nucleotide sequence of SEQ ID No. 38. Once expressed, the LTB
sequence would self assemble into a pentamer arid associate, non-covalently, with the CS3-chimera to form a hetero-hexarneric holotoxin-like structure, [0084] CS6 toxin chimera examples are also illustrated in Table 5. For CS6 chimeras, as iri CS3, one or more CS6 firnbrial subunits are connected via a polypeptide linker, preferably a tetrapeptide or triglycine. The C-terminal most CS6 fimbrial subunit is then connected to a donor 0 strand, via a polypeptide linker. The donor strand can be homologous or heterologous to the C-terminal fimbrial subunit. The donor strand is then connected to a toxin component (e.g., CTA2). .in a preferred embodiment, like fbr CS3, the chimera is co-expressed, with LTB, which self assembles into a pentamer to form a non-covalent association with the chimeric adhesion-toxoid fusion molecule [0085] Although many additional combinations are possible, in the examples shown in Table 5, the constructs are diallers of CS6 subunits, connected via a tetrapeptide linker, with the C-terminal fimbrial subunit connected, via a tetrapeptide linker to a donor strand. The donor strand can be homologous or heterologous to the C-terminal most fiinbrial subunit. However, in the examples in Table 5 the donor strands are heterologous to the C-terminal fimbrial subunit. The donor strand is then connected to a cholera toxin A2 (CTA2) subunit, The polypeptide sequences of one of the examples is as in SEQ ID
No. 43, which is encoded by the nucleotide sequence of SEQ ID Nos. 42. :In this example, the N-terminal subunit is CssA, with the N-terminal 15 amino acids of the mature CssA sequence deleted, In this example, a pelB leader sequence (22 amino acids) was also added, which is illustrated in FIG. 14.
Example 3: C fejuni capsule polysaccharides [0086j Recent development of a molecular CPS typing system re-enforced the strong correlation between CPS and Penner types (Poly, et al., T. Clin. Microbiol.
49: 1750 (2011)). Both Penner serotyping and molecular CPS typing have revealed the predominance of a handful of CPS types worldwide, Also, despite over 60 Penner serotypes having been identified, most Campylobacter diarrhea] disease is caused by C.
jejuni expressing only a limited number of serotypes. Therefore, only selected strains of C. jejuni, predicated on epidemiological studies, provides suitable candidate strains for development of vaccine compositions. However, despite the importance of this organism to human disease, there are no licensed vaccines against C. jejuni .
[0087] C. jejuni capsule polysaccharide (CPS) was extracted from C. jejuni strains selected based on their association with diarrheal disease. CPS fi-om bacteria was extracted by hot water¨phenol extraction for 2 h at 70 C. The aqueous layer was dialyzed (1000 Da) against water followed by ultracentrifugation to separate the CPS
from the LOS. The supernatant material containing the CPS was subjected to size-exclusion chromatography (Sephadex G50) for further purification to yield the intact CPSs. Monosaccharide composition was performed using a procedure amenable to the alditol acetate method (Chen, et al., Carbohydr. Res. 343: 1034 (2008)) with the alditol acetates 'being analyzed in a ThennoFinniga.n POLARISTm-Q (Thermo Fisher Scientific, Inc, 'Waltham, MA) gas chromatographimass spectrometer (GC/MS) using a DB-17 37' capillary column. The sugar linkage types were characterized by characterization of the permethylated alditol acetates by GC/MS as previously described (Chen, et al., Carbohydr. Res. 343: 1034 (2008)), The NMR experiments were perfomied on a Bruker 400 Mfiz spectrometer (Bniker Corporation, Billeria, MA) equipped with a Bruker cryo platform at 295 K with deuterated trimethylsilylpropanoic acid and orthophosphoric acid as external standards. The structures of important pathogenic C. jejuni capsule polysaccharides are shown in Table 6.

Table 6 Capsule Polysaccharide structure tvoe Hsi I

[MeOPN]¨*3)-Fnif Frnf-(3,¨[MeOPN1 HS44 --42)-Gro-( 1 HS3 ¨44)4P¨,61-alpha-D-Gal-( 1 ¨,3)-[P---2/7]-6-d-aipha-D-ido-tiep-(1¨; or ¨1=41)4P---4 31-alpha-D-Gal-( 1 ¨6 )-[P-61-1,-glycero-alpha-D-ido-Hep-( 1-4 (where P represents 0-tnethyl-phosphoratnidate) HS4/13/64 ¨6)-6-deoxy-beta-D-ido-Heptose-(1---44)-beta-D-GicNAe-(1----0, HS3/36 ---43)-P-D-G loNAc-(1 HS15 [----,3)-a-Araj-1( 1 ¨6 )-6-d-a-guio-liepp-( 1 HS 10 ;

6-d-a-ga1-iep MeOPN
HS13 MeOPN

--------- 1¨Y4)-p-G1ce-( l -6-d-ct-ido-Hen$,:ijõ
HS13 M ethiN

L-4)-f1-Glop-( 1-43 HS2 [MeOPNI

(3 ,6,-O-Me)-D-g1yc ero-a-L-gle-Hepp [MeOPNI [MeOPNI

[0088] Additionally, the capsule polysaccharide from the HS5 strain of C.
jejuni can be attached, HS 5 contains a complex of variations of polysaccharides. These include the following structures:
NeOPIk,fr. NeOPNT'' 7 Is: 7 a-Dideoxy4-lep ca-Dideoxy-Hep A 6 v &6 7)a-DD--Hep( I 3)Glucito1(6 P 7)a-DD-I-Iep(1 2)0imitoi(6 P

deoxy-l-lep a-Dideoxy-Ilep a-Dideoxy-l-lep [MeOPN]-- [MeOP11' [MeOPNr`
ti 7)a-DD-1-lep( 7)c-t-DD-Hep(I ----> 3)Glucitol(6 1' --u-Dideoxy-liep a-Dideoxy-liep 7 s- 7 1, [MeOPNr- [MeOPNr [MeOPNT.-Exampk 4: induction of immune response by ETEC-Carnpylobacter capsule conjugates.
[0089] Induction of an immune response by the conjugates was evaluated. In these studies, BALM mice were immunized with escalating amounts of vaccines administered with alhydrogel (Sergent Adjuvants, Clifton, NJ). Mice received a total of two immunization at a 4-wee1 interval.
[0090] The results of these studies is illustrated in FIG. 4. As shown. in FIG. 4, two weeks following the first immunization, mice immunized with 1-1S36-CfaE13 (10 ptg, 60 /1g) and HS36-CfaE (60 pg) exhibited significant levels of serum IgG
antibodies specific against HS36 CPS (p< 0,05) (see FIG, 4 (A)), comparared to pre-immune sera.
Following two immunizations all groups of immunized animals had antibody levels that were significant increased (p<0.)5) compared to levels observed after only one immunization. This effect was not dose dependent at the vaccine doses tested.
[0091] Purthemore, as illustrated in FIG. 4 (B), antibodies against CfaE was also determined by ELISA. As shown in FIG. 4(B), all rnice immunized with the conjugate vaccine possessed significant levels of anti-CfaE IgG (p<0.05) for CPS ¨ CthE
or CPS
CfaEB. No dose dependent effects were observed and all groups displayed similar levels of CfaE-specific IgG.
[0092] The data shown in FIG. 4 illustrates that the conjugate vaccine comprising an ETEC adhesin-based carrier protein conjugated to a C jejuni CPS is capable of inducing an immune response against both bacterial components, i.e., C. jejuni CPS and ETEC
CfaE.
[0093] To detemtine the levels of functional anti-adhesive antibody generated by HS36 conjugate vaccines, serum samples were tested by hemagglutination inhibition assay (HAI) assay in order to measure functional anti-adhesive antibodies present in the immune mouse serum.
[0094] The HAI assays were conducted by evaluating samples from each animal.
The samples were initially diluted 1:8, then diluted two-fold over a wide range of dilutions.
Each serum dilution was incubated with an equal volume of CFAII+ ETEC bacteria (strain H104)7), whith further diluted the senun 1:2. The final lowest dilution tested dwas 1:16, which was the limit of detection (Lt). The pre-incubated mixture was . 4.1 subsequently mixed with bovine erythrocytes in the presence of 0.5c.!4) mannose in bottom 96-we11 plates, In the absence of anti-adhesive antibodies, the erythrocytes formed visible agglutinated "buttons" of cells. In the presence of anti-adheisve antibodies, agglutination was inhibited. The :HAI titer was the highest serial dilution that completely inhibited agglutination. If there was not detectable inhibition at the lowest serum dilution of 1:16, the samples were assigned a value of one-half of the detection limit (i.e., 8) for computational purposes.
[0095] The results of the HAI analysis are illustrated in FIG, 5. Prior to immunization, pooled serum contained HAI titers below the the assay's level of detection, Following immunization, all groups of mice displayed significantly (p<0.05) higher levels of anti-adhesive antibodies in their sera compared to pre-immune titers. Genrally, mice immunized with HS36-CfaEB conjugate vaccine exhibited higher HAI titers, However, the only significance difference (P<0,05) observed was between mice immunized with an HS36-Cfa.EB (60 pg) and HS36-CfaE (10 Exarnpk 5: Inunune response against Inultiple MeOPN-6-Gal Synthesis of polysaccharide construct [0096] A polysaccharide constructed was synthesized as shown in FIG. 6.
Starting from a previously reported compound 4-methoxyphenyl-ct-D-galactopyranoside (see also FIG.
7, structure 1) (Comfort, et al., Biochena. 46: 3319-3330 (2007)), trityl group was selectively introduced to C-6. Originally, benzoylation was performed on compound (FIG. 7, structure 2), however extensive migration observed during the introduction of MeOPN lead us to look for a more suitable protecting group. Therefore, ally' groups were selected to protect the C-2, C-3 and C-4 positions which were resistant to migation. Allyl groups were later deprotected with catalytic hydrogenolysis which proved to be compatible with the MeOPN modification.
[00971 As shown in FIG. 6 and FIG. 7, after allyl groups were installed, an amino-pentanyl linker was introduced to the anomeric position as a site for conjugation. Starting from galactoside (FIG. 7, structure 3), 4-methoxyithenyl group (GP) was first removed with cerium ammonium nitrate (CAN). The corresponding hemiacetal was then converted into trichloroacetimidate donor. 5-Amino-N-phtha1imido-pentany1 linker was then introduced with TI SOTf as activator at 0 'C. Compound 5 (FIG. 7) was collected with 65{?.,' as the (3 anomer and 29% as the a anomer. The removal of trityl group gave a free 6-hydroxyl goup for modification.
[00981 The strategy for the introduction of MeOPN group was inspired by a similar reaction initially proposed by C. Mara et al, Bloom. Med. Chem. Lett. 6180-6183 (2011).
Compound 6 (FIG. 6 and FIG. 7) was treated with commercially available methyl dichlorophosphate in the presence of triethyl amine, followed by ammonolysis.
Due to the chirality nature of the newly introduced MeOPN (R and S), product 7 (FIG.
7, structure 7) was collected as a mixture of two diastereoisorners. H NMR was able to confirm that product '7 (FIG. 7) was indeed a 1:1 mixture of two diastereoisomers, revealing two sets of signals throughout the spectrum, such can be seen for anomeric and 0-Me signals. The reaction yielded a mixture of side products, the most abundant being the 0-Me group being replaced by a second NH2, accounting for the poor yield of this reaction.

[0099] Allyl and phthlimido protecting groups were removed with palladium (II) chloride and hydrazine respectively, generating product 9 (FIG. 7, structure 9).
Similar to compound 7 (FIG, 7), a mixture of diastereoisomers is apparent in NR. Although not optically pure, the 3IP NMR result agrees with native MeOPN-containing polysaccharides, having a phosphorous signals around 14 ppm.3 IH-311) HC NR
experiment was able to confirm that theMeOPN was introduced to the 0-6 position, showing correlation signal with 0-Me as well as the H-6 signals.
Induction of immunity against IvleOPN-6-Ga1 [00100] In one embodiment, galactose modified at the 6 carbon with 0-methyl phosphorarnidate (MeOPN-6-Gal) is used to induce immunity against multiple C.
jejuni strains, even those strains not expressing MeOPN-6-Ga1, As illustrated in FIG.
8, the monosaccharide construct MeOPN-6-Gal was recognized by antibody against capsule polysaccharide isolated from HS23/36, conjugated to CRM197. Unexpectedly, antibody against polysaccharide from HS, conjugated to CRMi97, also elicited an equivalent response, as anti-HS23/36 CRMI97 conjugate, against Me0PN-6-Ga1. Also, anti-HS
I-CRMI97, also reacted to MeOPN-6-Ga1, although to a somewhat less extent.
[00101] The strong cross-reactivity with N4e0PON-6-Ga1 exhibited against and HS 4 antibody may be explained by the the fact that MeOPN-6-Ga1 share epitopic structures with 1-S23/36 and IS' c,apsule polysaccharides. One explanation may be that the MeOPN group in both HS23/36 and HS 4 is to a primary hydroxyl. The cross reaction of MeOPN-6-Ga1 (HS23/36) with HS, which contains MeOPN-7-6d-13-1-ido-Heptose, was unexpected, but may be due to the linkage of MeOPN to primary hydroxyl positions on both sugars. This feature is illustrated in FIG 8 by the arrow.
Example 6: immunogenic composition against C. jejuni and enterotoxigenic Escherichia coli (ETEC) using a combined C. jejuni capsule/ETEC construct [00102] A synthetic conjugate vacci.ne strategy can be developed to protect against multiple enteric pathogens. Most efforts at development of vaccines against bacterial enteric pathogens are limited to a specific pathogen. The ability to combine vaccines against multiple, antigenically variable pathogens in a single, multi-valent, injectable .vaccine would greatly simplify approaches to prevent acquisition and transmission of these pathogens worldwide. Globally, ETEC and C. jejuni are among the leading causes of bacterial diarrheal disease. in addition CJ has been causally linked to several serious sequelae including Guillain Barre Syndrome, irritable bowel syndrome, and reactive arthritis. Moreover, recent studies have indicated an association between CJ
infections and malnutrition and growth stunting in. young children in resource-limited settings.
[00103] Using conventional methods, we have developed conjugate vaccines containing CI polysaccharide capsules that have proven to be immunogenic in multiple animal species and to confer protection against C jejuni diarrhea in NHP. The newer synthetic approach is based on recent data that the imrnunodominant epitope on CJ
polysaccharide capsule conjugate vaccines is the MeOPN modification found on different sugars in different capsule types.

[001041 Therefore, an immunogenic platform against both C. ,i.c_juni and ETEC
can be created by linking synthetic MeOPN-sugars to different ETEC protein antigens.
The approach could also be extended to include Shigella lipopolysaccharides (synthetic or detoxified) conjugated to ETEC proteins. Thus, this platform could form the basis of a multivalent vaccine against three major bacterial diarrheal pathogens.
Conjugation can also serve as a protein carrier to enhance immunogenicity of the Campylobacter construct.
[00105] It is envisioned to conjugate the construct of Examples 3 - 5 to an ETEC
construct. The overall method of conjugating includes oxidizing C jejune CPS, for example, with NaI04 in sodium acetate (pH 4.0), Oxidized CPSs were desalted with a 5 kDa cutoff membrane by stirred ultrafiltration, which is subsequently lypholized. ETEC
proteins are then added. The stoichiometery protein to CPS can vary, however, a typical ratio is 1:2 protein to CPS by mass. The concentration of components can be by any method. However, for example, polysaccharide concentration was determined by antrhone assay and protein concentration was determined by Pierce 660 protein assay or the BCA assay. NaCNBI-13 is then added. The conjugates can then be subsequently desalted by ultrafiltration and lyophilized. CPS, ETEC proteins and conjugates were analyzed, for example by SEC-HPLC or by SDS polyacrylaminde gel electrophoresis (PAGE), or other methods, .Example 7: Non-human primate response [00106] The immunogenicity of CfaE-HS23/36 and CB-S23/35 conjugates was observed in mice, as well as induction of hemagglutination inhibition (HAI) titers against Cal in mice. The amino acid sequence of the dscCfaE construct used is SEQ ID
No.
138 (nucleotide sequence is SEQ ID No. 139). The dsci9CfaE amino acid sequence is SEQ ID No. 143 (nucleotide sequence is SEQ ID No. 142). The amino acid sequence for dsci,ClaEB is SEQ ID No. 141 (nucleotide sequence is SEQ ID No. 140).
[00107] The CfaEB-HS23/36 conjugate was down-selected in order to proceed to studies in Aottis nancyrnaae. This non-human primate (NHP) model was selected because it has been used as a diarrhea' disease model for both E'I'EC and C.
jejuni. We synthesized a lot of the CfaEB.IS23/36 vaccine that was sufficient in size for three N-IP
studies by reductive amination. The first such study, which is the only one that has been completed, was a dose finding study followed by a C. jejuni challenge.
[00108] The desiga of this NHP study is shown in Table 7. Animals (6 per group) were immunized three times at days 0, 42, and 84. The CfaEB-HS23136 vaccine was given subcutaneously at either 0.5 us or 3.5 ug polysaccharide (PS) adjuvanted with aluminum hydroxide. The ratio of PS to protein in the vaccine was roughly 1:1 so this was equivalent to 0.5 or 3.5 ug of CLEB per dose. The 3.5 ug dose was also given intradermally (ID) with poly-IC as adjuvant. This was done to bridge to previous work done using ID immunizations with CfaEB alone. Similarly, another group was given 11S23/36-CR1\4197 subcutaneously to bridge to previous work with the same capsule conjugated to another protein. Finally, the control group was immunized with PBS. On day 148 the animals were all challenged with 4 x 1011 CPU of CG8421, an strain.

Table 7: Design of NHP study Group Route CfuEB- CPS- Mont (ug) Poly IC PBS
CPS CRAI197 (110 1 SC 0,5 300 2 SC 1 3,5 300 3 ID 1 3.5 ................. 1 100 4 1 SC 3.5 SC

[00109] Animals were observed for diarrhea disease daily for 10 days following challenge. Dian-hea was defined as two or more days of consecutive of stools that were grade 3. The results are summarized in Table 8. Only 3/5 animals in the PBS
group developed diarrhea for an attack rate of 60%. Note that one animal was eliminated because it developed diarrhea prior to challenge. The mean time to onset of disease in this negative control group was 2.3 days and the mean duration of illness was 5.3 days.
The attack rate in the animals immunized with the FIS23/36-CR1197 vaccine was 33%
(2/6), with a mean onset of disease of 2 days and a mean duration of illness of 4 days (45% efficacy). Animals that were immunized with CfaEB-HS23/36 intraderrnally with poly IC also showed an attack rate of 33% with a mean onset of 1.5 days and a duration of two days (45% efficacy). The animals immunized subcutaneously with CfaEB-CPS
showed between 67-100% efficacy against diarrheal disease. The attack rate in the group immunized with 0.5 ug of the vaccine was 0 ((115, with one animal that vomited after challenge being eliminated) and the attack rate in the group im3munized with 3,5 ug of the vaccine was 20% (1/5, with one animal being eliminated due to diarrh.ea prior to challenge). The single animal in this group that did develop diarrhea had a later onset of disease (day 9). There were no significant differences among the control group and any of the im_munized animals due to the small numbers of animals per group, Table 8. Results of challenge with C. jejtmi CG8421.
Group Vaccine #iiiitotal Attack rate Mean days to Mean days of I Protective %) onset of illness efficacy diarrhea I (range) (re stg.t) CfaEB-CPS WS* 0 0 0 00 ¨
(0.5 ug) alum 2 CfaE-CS 1.15** 20 9 67 I (3.5 ug) alum --3 CfaEB-CPS 216 33 1.5 (l-4) 2 (2-4) 45 (3.5 ug) ply IC
4 CRM-CPS 2/6 33 2(-3) '(2-6) 45.
alum PBS 1:5"
60 23t1.6) = 5.3 (2-10) L
* One animal vorinted after challenge and was excluded *9 Animals were excluded from analyses due to diarrheal onset prior to challenge [(l 10] Serology results are shown in Fig. 9. Immune responses to CPS and to CfaE
were measured by :ELISA. Animals in groups I, 2 and 3 displayed IgG responses to both antigens and and IgA response to CfaE. Hemagglutination inhibition (HAI) titers against ETEC strain H10407 expressing CfaI firrihriae were determined and are shown in Fig. 10.
The results indicate that HAI titers were detected in animals in groups I, 2 and 3, with group 2 showing the highest titers.
Example 8: Synthesis and immunagenicity of additional combinations of ETEC--Campylobacter capsule conjugates [00111] CssBA-I4S3 vaccine. CssBA is a recombinant form of the two subunits of that are fused together. This protein was conjugated to capsule from an HS3 strain by TEMPO oxidation. The conjugates were analyzed by SS-PAGE and immunoblotting.
Purified CssBA has a predicted 1\ilf of 31.8 kDa. The conjugate of CssBA-HS3 CPS runs as two bands, one slightly smaller than CssBA and one that runs at approximately 60 kDa. The bands in the conjugate react with both anti-CssB.A antiserum arid antibodies to whole cells of HS3, indicating that polysaccharide has 'been conjugated to the protein.
[00112] Mice were immunized subcutaneously with three doses of the vaccine given at 4 week intervals. Doses were 5 ug by weight or 25 'ug by weight. Animals were bled at day 0 and two weeks after each immunization and the response to CssBA and to CPS
were determined by ELISA. The results, shown in Fig. 11, indicate that there was a robust response to both the protein and the polysaccharide at both doses.
[0011.3] 1,,TB-11S4 vaccine. LTB is the binding component of the heat labile enterotoxin of ETC, Recombinant LTB, which is not toxic, was conjugated to the polysaccharide capsule of an HS4 strain by reductive amination. The conjugate was analyzed by irnmunoblotting as shown in Fig. 12. Immunodetection with anti-LTB

antiserun-i revealed a single band for LTB at approximately 10 lc:Da. The conjugate contained 4 major bands ranging from ¨20kDa->75kDa that were reactive with both anti-LTB and anti-HS4 antiserum, indicating successful conjugation.
[00114] Mice were irm-nunized with three doses of either 5 or 25 ug (by weight) of the LTB-HS4 conjugate subcutaneously at 4 week intervals and the serum immune response was detenrnined. The results, shown in Fig. 12, indicate that there was a robust immune response to both the H54 capsule and to LTB at both doses.
Example 9: Conjugation to Shigella lipopolysaccharide (LPS) [00115] There are four species of Shigella, a human pathogen cause diseases such as diarrhea and bacilliary dysentaery: Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei are important enteropathogens Strains of Shigella spp. Express long-chain lipopolysaccharides. The chemical structures for many strains has been determined (see Liu, et al., FEMS Microbiol. Rev. 32: 627-653 (2008)).
[00116] An object of this invention, is a Shigella LPS-ETEC construct. The construct comprises an ETEC construct, as the above examples, conjugated, to a Shigelic,? LPS, as an alternative or in addition to C jejuni capsule polysaccharide. ft is envisioned that any of the Shigella spp. can be conjugated to the ETEC construct. As an example, the Shigella flexneri 2a ITS is illustrated, as a potential LPS structure that can be conjugated to an ETEC construct, as follows:
a-D-Glc(1-4) -2)-a-L-Rhap-(1-2)-a-L-Rhap-(1-3)-a-L-Rhaip-( I -3)-P-D-G1cNAcp(1-.

Claims (25)

What is claimed is:

1. A multi-agent immunogenic construct, comprising a Campylobacter jejuni capsule polysaccharide conjugated to a protein carrier, wherein said protein carrier comprises an Escherichia coh enterotoxigenic recombinant polypeptide construct.

2. The multi-agent immunogenic construct of claim 1, wherein said Escherichia coil recombinant polypeptide construct comprises a minor or major subunit connected to one or more major fimbrial subunits or immunogenic fragments, thereof, of the same timbrial type, via a polypeptide linker, and wherein each of the one or more major fimbrial subunits contain a donor .beta. strand and are also connected to each other via a polypeptide linker wherein the C-terminal Escherichia coli fimbrial major subunit is connected, via a linker, to a C-terminal donor .beta. strand derived from a major Escherichia coli fimbrial subunit that is homologous or heterologous to the immediately N-terminal major subunit, or wherein said recombinant polypeptide construct is connected to one or more additional recombinant polypeptide constructs, wherein each of the constructs contain fimbrial subunits derived from a different fimbrial type than any of the other constructs and wherein the the recombinant polypeptide construct can contain a C-terminal histidine tag at the C-terminus.

3. The multi-agent immunogenic construct of claim 1, wherein the multi-agent immunogenic construct comprises Shigelia lipopolysaccharide in place of or in addition to the campylobacter jejuni capsule polysaccharide.

4. The multi-agent immunogenic construct of claim 1, wherein said molar ratio of Campylobacter jejuni or Shigelia spp, lipopolysaccharide to Escherichia coil recombinant protein carrier is 1:1 to 5:1.

5. The multi-agent immunogenic construct of claim 2, wherein said minor or major Escherichia coli fimbrial subunits are derived from ETEC strains selected from the group consisting of Class 5, CS3 and CS6.

6. The mufti-agent immunogenic construct of claim 2, wherein the Escherichia coli recombinant polypeptide construct comprises an amino acid sequence selected from the group consisting of SEQ ID Nos, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 102, 103, 105, 107, 109, 112, 114, 138, 141 and 143,

7, The multi-agent immunogenic construct of claim 2, wherein the Escherichia call recombinant polypeptide construct is encoded by the nucleotide sequence of SEQ
ID
Nos. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 42, 101, 104, 106, 108, 110, 111, 113, 139, 140, and 142.

8. The mufti-agent immunogenic construct of claim 2, wherein said Campylobacter jejuni polysaccharide is a repeating trisaccharide structure having the formula selected from the group consist of:
[.fwdarw.3)-alpha.-D-Gal-(1.fwdarw.2)-6d-alpha-D-altro-Me-Hep-(1.fwdarw.3)-.beta.-D-GlcNAc-(1.fwdarw.]n ;
[.fwdarw. 3)-.beta.-6-deoxy-D-ido-Heptose (1 .fwdarw. 4)-.beta.-D-GlcNAc-(1.fwdarw.]n ;
[.fwdarw.3)-.alpha.-Ara.function.-(1.fwdarw.3)-6-d-.alpha.-gulo-Hepp-(1.fwdarw.]n ;
[.fwdarw.43)-L-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1.fwdarw.]n, with non-stoichiometric substitution of O-methyl-phosphoramidate at position 2 of L-glycero-beta-D-ido-beptose;

[.fwdarw.3)-6d-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1.fwdarw.]n, derived from HS13, with non-stoiehiometric substitution of O-methyl-phosphoramidate at position 2 or/and 7 of 6-deoxy-beta ¨D-ido-heptose;
[.fwdarw.3)-L-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1.fwdarw.]n ;
[.fwdarw.3)-L-alpha-D-ido-Hep-(1->4)-alpha-Gal-(1.fwdarw.]n, with non-stoiehiomoetrie substitution O-methyl-phosphoramidate at position 2 of 6-deoxy-alpha-D-ido-heptose;
; and [.fwdarw. 3)-6d-alpha-D-ido-Hep-(1->4)-alpha-Gal-(1.fwdarw.]n, derived from HS3, HS13 and HS50 with non-stoichiometric substititution of O-methyl-phosphoramidate at position 2 of L-glycero-alpha-D-ido-heptose, wherein "n" is 1 to 100,

9. The multi-agent immunogenic construct of claim 3, wherein said Shigella lipopolysaccharide has the structure:

10. The multi-agent immunogenic construct of claim 2, wherein said donor .beta. strand contains 12 to 16 amino acids.

11. The multi-agent immunogenic construct of claim 2, wherein said N-terminus of said minor or major subunit contains an 18-22 amino acid signal peptide.

12. The multi-agent immunogenic construct of claim of claim 2, wherein the amino acid sequence of said polypeptide linker is the amino acid sequence of SEQ ID
No. 5 or a tri-glyicine.

13. The multi-agent immunogenic construct of claim of claim 2, wherein one or more major subunits contain a deletion of the 14 to 18 N-terminal amino acids.

14. The multi-agent immunogenic construct of claim 5, wherein said Escherichia coli fimbrial minor subunit is selected from the group consisting of CfaE, CsfD, CsuD, CooD, CsbD, CosD, CsdD, CotD and wherein said major subunit is selected from the group consisting of CfaB, CsfA, CsuA2, CooA, CsdA, CosA, CsbA, CotA, CstG, CstH, CssA, and CssB, or immunogenic fragements or derivatives, thereof.

15. The multi-agent immunogenic construct of claim 14, wherein said amino acid sequence of said Escherichia coli fimbrial minor subunit is selected from the group consisting of SEQ ID Nos. 45, 46, 51, 52, 57, 58, 65, 71, 75, 79, 83, 88, 90, 93, 95, and 97, or derivatives thereof, and wherein said amino acid sequence of said Escherichia coil fimbrial major subunit is selected from the group consisting of SEQ ID Nos, 2, 4, 48, 49, 54, 55, 60, 61, 63, 67, 69, 73, 77, 81, 85, 87, 89, 91, 92, 94, 96, 98, 99, 101, 135, and 137, or derivatives thereof.

16. A method of inducing an immune response against C. jejuni strains, wherein said method induces an immune response against one or more enterobacteria selected from the group consisting of C strains, Escherichia coli, and Shigella, comprising the steps:
a. administering the multi-agent immunogenic composition of claim 2 at a dose range of 0.1 µg to 10 mg per dose;
b, administering a boosting dose of said capsule polysaccharide composition at a dose range of 0.1 µg to 10 mg per dose.

17. The method of claim 16, wherein said Campylobacter jejuni capsule polysaccharide comprises the polysaccharide structures of claim 8.

18. The method of claim 16, wherein said multi-agent immunogenic composition comprises the construct of claim 3.

19. The method of claim 16, wherein said Escherichia coli enterotoxigenic recombinant polypeptide construct wherein said Escherichla coli fimbrial minor subunit is selected from the group consisting of SEQ ID Nos. 45, 46, 51, 52, 57, 58, 65, 71, 75, 79, 83, 88, 90, 93, 95, and 97, or derivatives thereof, and wherein said amino acid sequence of said Escherichia coil fimbrial major subunit is selected from the group consisting of SEQ ID Nos. 2, 4, 48, 49, 54, 55, 60, 61, 63, 67, 69, 73, 77, 81, 85, 87, 89, 91, 92, 94, 96, 98, 99, 101, 135, and 137, or derivatives thereof.

20. The method of claim 16, wherein the Escherichia coil recombinant polypeptide construct comprises an amino acid sequence selected from the group consisting of SEQ
ID Nos, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 102, 103, 105, 107, 109, 112, 114, 138, 141, and 143,

21. The method of claim 16, wherein the Escherichia coli recombinant polypeptide construct is encoded by the nucleotide sequence of SEQ ID Nos, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 42, 101, 104, 106, 108, 110, 111, 113, 139, 140, and 142.

22. The method of claim 16, wherein said donor 3 strand contains 12 to 16 amino acids.

23. The multi-agent immunogenic construct of claim 16, wherein said N-terminus of said minor or major subunit contains an 18-22 amino acid signal peptide.

24. The multi-agent immunogenic construct of claim 16, wherein the amino acid sequence of said polypeptide linker is the amino acid sequence of SEQ ID No. 5 or a tri-glyicine.

25. The multi-agent immunogenic construct of Claim16, wherein one or more major subunits contain a deletion of the 14 to 18 N-terminal amino acids.